Brassica oleracea plants with improved nutritional value

ABSTRACT

The invention provides compositions and methods relating to the elevation of glucoraphanin compared to standard  Brassica oleracea  varieties. The invention also relates to the production of hybrid varieties having desired glucosinolate contents. The invention further provides plants, plant parts, and seeds comprising such traits and comprising a Myb28 allele from  Brassica villosa  that is not genetically linked to an ELONG allele from  Brassica villosa.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. provisional application No.61/700,762, filed Sep. 13, 2012, herein incorporated by reference in itsentirety.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“SEMB008US_ST25.txt”, which is 8 kilobytes as measured in MicrosoftWindows operating system and was created on Aug. 28, 2013, is filedelectronically herewith and incorporated herein by reference.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to the development and use of Brassicaoleracea plants with a recombined chromosomal segment.

2. Description of Related Art

Glucosinolates are allelochemicals present in 16 families of plantspecies, especially in Brassicaceae, of which broccoli is a notableexample. Although there are over 120 different glucosinolates identifiedin nature, closely related taxonomic groups typically contain only asmall number of such compounds. Glucosinolates that are dominant inbroccoli such as glucoraphanin and glucoiberin are derived biochemicallyfrom the amino acid methionine. In the glucosinolate pathway, methionineis converted to homo-methionine and dihomomethionine by the activity ofthe ELONG (elongation) locus by adding a single carbon unit to the taileach time. Homo-methionine is eventually converted to 3-methylthiopropylglucosinolate (glucoiberin; “MSP”) while dihomomethionine is convertedto 4-methylthiobutyl glucosinolate (glucoraphanin; “MSB”). Theseglucosinolates (glucoiberin and glucoraphanin) are potent inducers ofphase II detoxification enzymes, such as glutathione-S-transferase andquinone reductase, which promote the metabolism and excretion ofpotential carcinogens.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a Brassica oleracea plantcomprising a Myb28 allele from Brassica villosa and lacking an ELONGallele from Brassica villosa genetically linked to said Myb28 allele,wherein the Myb28 allele confers elevated glucosinolates when comparedto a plant that lacks the Myb28 allele.

In one embodiment, the plant is a broccoli plant. In other embodiments,the plant is inbred or hybrid. In another embodiment, the plant ishomozygous for said Myb28 allele from Brassica villosa. In yet anotherembodiment, the plant is heterozygous for said Myb28 allele fromBrassica villosa. In still another embodiment, the ELONG allele is fromBrassica oleracea.

In another aspect, the present invention provides a part of a plant ofthe invention. In some embodiments, the plant part may further bedefined as a leaf, an ovule, a floret, pollen, a head, or a cell.

In yet another aspect, the present invention provides a seed thatproduces a plant of the invention.

In still another aspect, the present invention provides a Brassicaoleracea plant comprising a chromosomal segment that comprises a Myb28allele from Brassica villosa and lacking an ELONG allele from Brassicavillosa genetically linked to said Myb28 allele, wherein the segmentconfers elevated glucosinolates relative to a plant lacking the Myb28allele, and wherein a sample of seed comprising the chromosomal segmentwas deposited under ATCC Accession Number PTA-13165. In one embodiment,the invention provides a seed that produces such a plant. In anotherembodiment, the invention provides a plant part, wherein the part is aleaf, an ovule, a floret, pollen, a head, or a cell. In anotherembodiment the plant is a B. oleracea plant.

In another aspect of the present invention a recombined DNA segmentcomprising a Myb28 allele from Brassica villosa and an ELONG allele fromBrassica oleracea is provided. In one embodiment, the DNA segment isfurther defined as comprised within a cell. In another embodiment, theDNA segment is further defined as comprised within a seed. In yetanother embodiment, the DNA segment is further defined as comprisedwithin a plant.

In another aspect, the present invention provides a method for obtaininga Brassica plant comprising a desired glucosinolate compositioncomprising: a) obtaining a Brassica plant heterozygous for a Myb28allele from Brassica villosa that confers elevated glucosinolates and isgenetically linked in the plant to an ELONG allele from Brassicavillosa; (b) obtaining progeny of the plant; and (c) selecting at leasta first progeny plant in which recombination has occurred such that theprogeny comprises the Myb28 allele but not the ELONG allele fromBrassica villosa, wherein the progeny plant possesses a desiredglucosinolate composition as a result of the presence of the Myb28allele but not the ELONG allele from Brassica villosa.

In one embodiment, selection of the progeny plant comprises identifyinga progeny plant that (1) comprises a genetic marker genetically linkedto the Myb28 allele in Brassica villosa and/or lacks a genetic markerpresent at the corresponding locus in said Brassica plant, and (2) lacksa genetic marker genetically linked to the ELONG allele from Brassicavillosa and/or comprises a genetic marker present at the correspondinglocus from said Brassica plant.

In another embodiment, selection of the progeny plant comprisesdetecting a polymorphism that is found in the genome of said plantflanked by the complements of SEQ ID NO:1 and SEQ ID NO:2. In a furtherembodiment, such allele(s) are detected by a PCR-based method usingoligonucleotide primer pair(s). In another embodiment, selection of theprogeny plant comprises detecting a polymorphism in said progeny plantthat is shown in FIG. 5. In a further embodiment, the Brassica plant maybe a B. oleracea plant.

In yet a further aspect, the invention provides a plant produced by amethod of the invention or a progeny thereof comprising the Myb28 allelebut not the ELONG allele from Brassica villosa. In one embodiment, theinvention provides a part of such a plant. In another embodiment, thepart of the plant is selected from the group consisting of a cell, aseed, a root, a stem, a leaf, a head, a flower, and pollen.

In another aspect, the invention provides a method for producing ahybrid Brassica oleracea plant with elevated glucosinolate contentcomprising crossing a first Brassica oleracea parent plant with a secondBrassica oleracea plant of a different genotype, wherein the firstparent plant comprises a Myb28 allele from Brassica villosa that lacksan ELONG allele from Brassica villosa genetically linked to said Myb28allele, wherein the Myb28 allele confers elevated glucosinolatesrelative to a plant lacking the Myb28 allele. In one embodiment, themethod further comprises producing a plurality of hybrid Brassicaoleracea plants comprising crossing the first Brassica oleracea parentplant with a plurality of second Brassica oleracea plants of differentgenotypes.

In still another aspect, the invention provides a method of producing aBrassica oleracea plant with a desired elevated glucosinolate contentcomprising introgressing into the plant a chromosomal segment comprisinga Myb28 allele from Brassica villosa and lacking an ELONG allele fromBrassica villosa genetically linked to said Myb28 allele, whereinsegment confers a desired glucosinolate content relative to a plantlacking the segment, wherein a sample of seed comprising the chromosomalsegment is deposited under ATCC Accession No. PTA-13165.

The term “about” is used to indicate that a value includes the standarddeviation of error for the device or method being employed to determinethe value. The use of the term “or” in the claims is used to mean“and/or” unless explicitly indicated to refer to alternatives only orthe alternatives are mutually exclusive, although the disclosuresupports a definition that refers to only alternatives and to “and/or.”When used in conjunction with the word “comprising” or other openlanguage in the claims, the words “a” and “an” denote “one or more,”unless specifically noted. The terms “comprise,” “have” and “include”are open-ended linking verbs. Any forms or tenses of one or more ofthese verbs, such as “comprises,” “comprising,” “has,” “having,”“includes” and “including,” are also open-ended. For example, any methodthat “comprises,” “has” or “includes” one or more steps is not limitedto possessing only those one or more steps and also covers otherunlisted steps. Similarly, any plant that “comprises,” “has” or“includes” one or more traits is not limited to possessing only thoseone or more traits and covers other unlisted traits.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and any specificexamples provided, while indicating specific embodiments of theinvention, are given by way of illustration only, since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the head from hybrid broccoli varieties Ironman (left)and RX05991199 (right). Varieties were grown in Summer at a spacing of50×50 cm.

FIG. 2 shows a horizontal cross section of stems from hybrid broccolivarieties Ironman (left) and RX05991199 (right). Varieties were grown inAutumn at a spacing of 50×50 cm.

FIG. 3 shows a profile of the head and stem from hybrid broccolivarieties Ironman (left) and RX05991199 (right). Varieties were grown inAutumn at a spacing of 50×50 cm.

FIG. 4 shows assay results for different ELONG alleles with theQTL1-BoGLS-ELONG marker. The V allele is the B. villosa alleleassociated with Myb28, which results in high 3-MSP/4-MSB ratio and hightotal glucosinolates. The A, B and C alleles are examples of allelesfound in broccoli without a B. villosa ELONG allele.

FIG. 5 shows an alignment between a consensus sequence of the Myb28locus from B. villosa contained in broccoli variety FT69, and aconsensus sequence of the corresponding locus from broccoli withoutincreased level of glucosinolate, e.g. B. oleracea, (Oleracea) (SEQ IDNOs.:8-9).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods and compositions relating to plants,seeds and derivatives of Brassica oleracea plants comprising a newrecombined introgression from Brassica villosa capable of conferringelevated 4-methylsulfinylbutyl glucosinolates (MSB), also known asglucoraphanin. It was also surprisingly found that plants comprising theintrogression were capable of consistently producing hybrids thatexhibited elevated glucoraphanin content relative to glucoiberin, whileresults were substantially more variable when using a parent linecomprising the non-recombined introgression, such as in the case of theMyb28 donor parent of the hybrid PS05151639, which comprises Myb28 andELONG alleles from Brassica villosa (US Patent Appln Pub No.2011/0055945). The ability to produce multiple elite hybrid progeny withelevated glucoraphanin content and/or ratio of glucoraphanin toglucoiberin from a single inbred parent is significant in that thenumber of elite Brassica oleracea parent lines available to producehybrid varieties is limited. The reduced introgression thereforesubstantially increases the utility of a given inbred and allows, forexample, the production of multiple hybrids potentially adapted todifferent growing environments, end uses, or other criteria, each havinga desired glucoraphanin content.

The new “reduced introgression” comprising a Myb28 locus from Brassicavillosa and lacking the ELONG locus of Brassica villosa that has to datebeen genetically linked thereto is capable of consistently conferring inhybrids derived from a plant comprising the introgression elevatedglucoraphanin relative to glucoiberin. One aspect of the currentinvention thus concerns methods for obtaining a Brassica oleracea plantcomprising at least one such reduced introgression, wherein theresulting Brassica oleracea plant and/or progeny derived therefromdisplays a desired glucoraphanin content relative to a control plantlacking the introgression. The invention thus provides plants possessinga desired glucoraphanin content conferred by a reduced introgression ofthe invention. In certain embodiments, methods for obtaining such plantscomprise obtaining a Brassica oleracea plant heterozygous for a Myb28allele from Brassica villosa that confers elevated glucosinolates and isgenetically linked in the plant to an ELONG allele from Brassicavillosa, obtaining progeny from such a plant, and selecting one or moresuch progeny plants wherein genetic recombination has occurred such thatthe progeny comprises an Myb28 allele from Brassica villosa, but doesnot comprise an ELONG allele from Brassica villosa. Such progeny orfurther progeny thereof may also possess a desired glucoraphanin contentas a result of the presence of the Myb28 allele but not the ELONG allelefrom Brassica villosa. In particular embodiments, the method maycomprise obtaining a progeny plant that comprises such allele(s) byidentifying one or more genetic markers genetically linked to the Myb28and/or ELONG allele(s). Identifying the genetic markers may comprise aphenotypic, a genetic, or a biochemical test, and may include screeninga parent and/or progeny plant for the presence of, for instance, one ormore allele described herein, including, for example, a Myb28 allelefrom B. villosa, an ELONG allele from B. villosa and an ELONG allelefrom B. oleracea.

Certain traits such as a glucoraphanin content relative to glucoiberincontent, or the unpredictability of the type of glucosinolates in hybridprogeny heterozygous for a Brassica villosa Myb28 allele, were found toco-locate with a glucosinolate trait in the Myb28 allele from Brassicavillosa and ELONG allele from Brassica villosa introgressions. Thus,formation of a “reduced” introgression is understood to be caused byrecombination event(s) in the vicinity of the Myb28 and ELONG QTL(s).Lines comprising a reduced introgression, i.e., which have undergone arecombination event close to the QTL having elevated glucosinolates mayefficiently be screened by use of molecular and/or phenotypic markers.Thus, plant populations or progeny of such populations, segregating(i.e., heterozygous) with respect to the QTL specified by Myb28 andELONG introgressions, may be screened for plants having a recombinantphenotype, e.g. elevated glucoraphanin levels relative to glucoiberinlevels.

In other embodiments, a method of the invention may comprise identifyinga Brassica oleracea plant comprising a Brassica villosa-derived reducedintrogression, and comprising a meiotic recombination between Myb28 andELONG alleles as described herein. In particular embodiments,identifying the introgression may comprise measuring glucoraphaninand/or glucoiberin using standard protocols. In certain embodiments, aplant of the invention comprising a reduced introgression as disclosedherein comprises an elevated average proportion of glucoraphaninrelative to glucoiberin compared to a plant comprising Myb28 and ELONGalleles from Brassica villosa, or a plant lacking a Myb28 allele fromBrassica villosa. In one embodiment, such a plant comprising an elevatedaverage proportion of glucoraphanin relative to glucoiberin is an inbredline, and in another embodiment is defined as a F1 hybrid having as oneor more parent a plant comprising a reduced introgression of theinvention. In particular embodiments, a plant of the invention isprovided comprising a ratio of glucoraphanin to glucoiberin of about10:1, 12:1, 15:1, 18:1, 20:1, 23:1, 25:1, 28:1, 30:1, 35:1 and about40:1. In one aspect, an increase in glucoraphanin content may becalculated in reference to a standard Brassica oleracea variety, such asthe broccoli variety Ironman.

A. Breeding of Brassica oleracea Lines Displaying Elevated Glucoraphanin

One aspect of the current invention concerns methods for crossing aplant comprising a reduced Myb28/ELONG introgression provided hereinwith itself or a second plant and the seeds and plants produced by suchmethods. These methods can be used for production and propagation ofcultivated Brassica oleracea plants displaying desired glucosinolatecompositions, including MSB and/or MSP content. Yet further, the plantsof the current invention having elevated glucosinolate content compriseimproved nutritional value of the plant relative to plants withoutelevated glucosinolates. The methods also can be used to produce hybridBrassica oleracea seeds and the plants grown therefrom. Hybrid seeds areproduced by crossing such lines with a second Brassica oleracea parentline. The hybrids may be heterozygous or homozygous for the reducedintrogression.

Brassica villosa is a wild species endemic to northwest and centralSicily, and thus a Myb28 allele could be obtained by one of skill in theart from a plant selected from the wild. Alternatively, Myb28 allelesare known in the art and may be obtained from other sources for use withthe invention, including SNR 347 (FT69; referred to as 428-11-69 inMithen et al., Theor Appl Genet, 106:727-734; 2003), BR384-014, SNP13(580333), SNP88 (BRM51-1210), BR384-020, B1639 (ATCC Accession NumberPTA-9676), BRM51-1162 (ATCC Accession Number PTA-9675) and RX 05991199(ATCC Accession No. PTA-13165). In accordance with the invention, aplant provided herein will generally lack an ELONG allele from Brassicavillosa genetically linked to the Myb28 allele. This can be achievedaccording to the invention through crossing a plant comprising Myb28 andELONG alleles from Brassica villosa with Brassica plants not comprisingthe Myb28 and ELONG alleles from Brassica villosa, including standardBrassica oleracea varieties. This includes the many broccoli varietieswell known in the art, among others.

The goal of vegetable breeding is to combine various desirable traits ina single variety/hybrid. Such desirable traits may include any traitdeemed beneficial by a grower and/or consumer, including greater yield,resistance to insects or disease, tolerance to environmental stress, andnutritional value. Breeding techniques used in an attempt to obtaindesired traits take advantage of a plant's method of pollination. Thereare two general methods of pollination: a plant self-pollinates ifpollen from one flower is transferred to the same or another flower ofthe same plant. A plant cross-pollinates if pollen comes to it from aflower of a different plant.

The development of uniform varieties requires the development ofhomozygous inbred plants, the crossing of these inbred plants, and theevaluation of the crosses. Pedigree breeding and recurrent selection areexamples of breeding methods that have been used to develop inbredplants from breeding populations. Those breeding methods combine thegenetic backgrounds from two or more plants or various other broad-basedsources into breeding pools from which new lines and hybrids derivedtherefrom are developed by selfing and selection of desired phenotypes.

In accordance with the invention, novel varieties may be created bycrossing plants of the invention followed by generations of selection asdesired and inbreeding for development of uniform lines. New varietiesmay also be created by crossing with any second plant. In selecting sucha second plant to cross for the purpose of developing novel lines, itmay be desired to choose those plants which either themselves exhibitone or more selected desirable characteristics or which exhibit thedesired characteristic(s) when in hybrid combination. Once initialcrosses have been made, inbreeding and selection take place to producenew varieties. For development of a uniform line, often five or moregenerations of selfing and selection are typically involved.

Uniform lines of new varieties may also be developed by way ofdoubled-haploids. This technique allows the creation of true breedinglines without the need for multiple generations of selfing andselection. In this manner true breeding lines can be produced in aslittle as one generation. Haploid embryos may be produced frommicrospores, pollen, anther cultures, or ovary cultures. The haploidembryos may then be doubled autonomously, or by chemical treatments(e.g. colchicine treatment). Alternatively, haploid embryos may be growninto haploid plants and treated to induce chromosome doubling. In eithercase, fertile homozygous plants are obtained. In accordance with theinvention, any of such techniques may be used in connection with a plantof the present invention and progeny thereof to achieve a homozygousline.

Backcrossing can also be used to improve an inbred plant. Backcrossingtransfers a specific desirable trait, such as elevated glucoraphanin,from one inbred or non-inbred source to a variety that lacks that trait.This can be accomplished, for example, by first crossing a parent (A)(recurrent parent) to a donor inbred (non-recurrent parent), whichcarries the appropriate locus or loci for the trait in question. Theprogeny of this cross are then mated back to the recurrent parent (A)followed by selection in the resultant progeny for the desired trait tobe transferred from the non-recurrent parent. After five or morebackcross generations with selection for the desired trait, the progenyare heterozygous for loci controlling the characteristic beingtransferred, but are like the first parent for most or almost all otherloci. The last backcross generation would be selfed to give purebreeding progeny for the trait being transferred.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalvariety. To accomplish this, a single locus of the recurrent variety ismodified or substituted with the desired locus from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphologicalconstitution of the original variety. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross; one ofthe major purposes is to add some commercially desirable trait to theplant. The exact backcrossing protocol will depend on the characteristicor trait being altered to determine an appropriate testing protocol.Although backcrossing methods are simplified when the characteristicbeing transferred is a dominant allele, a recessive allele may also betransferred. It may be necessary to introduce a test of the progeny todetermine if the desired characteristic has been successfullytransferred.

Brassica oleracea varieties can also be developed from more than twoparents. The technique, known as modified backcrossing, uses differentrecurrent parents during the backcrossing. Modified backcrossing may beused to replace the original recurrent parent with a variety havingcertain more desirable characteristics or multiple parents may be usedto obtain different desirable characteristics from each.

Many single locus traits have been identified that are not regularlyselected for in the development of a new inbred but that can be improvedby backcrossing techniques. Single locus traits may or may not betransgenic; examples of these traits include, but are not limited to,male sterility, herbicide resistance, resistance to bacterial, fungal,or viral disease, insect resistance, restoration of male fertility,modified fatty acid or carbohydrate metabolism, and enhanced nutritionalquality. These comprise genes generally inherited through the nucleus.

Direct selection may be applied where the single locus acts as adominant trait. Selection of Brassica plants for breeding is notnecessarily dependent on the phenotype of a plant and instead can bebased on genetic investigations. For example, one can utilize a suitablegenetic marker which is closely genetically linked to a trait ofinterest. One of these markers can be used to identify the presence orabsence of a trait in the offspring of a particular cross, and can beused in selection of progeny for continued breeding. This technique iscommonly referred to as marker assisted selection. Any other type ofgenetic marker or other assay which is able to identify the relativepresence or absence of a trait of interest in a plant can also be usefulfor breeding purposes.

Procedures for marker assisted selection are of particular utility forintrogression of given traits. Well known types of genetic markers thatcould be used in accordance with the invention include, but are notnecessarily limited to, Simple Sequence Length Polymorphisms (SSLPs),Cleaved Amplified Polymorphic Sequences (CAPs), Randomly AmplifiedPolymorphic DNAs (RAPDs), DNA Amplification Fingerprinting (DAF),Sequence Characterized Amplified Regions (SCARs), Arbitrary PrimedPolymerase Chain Reaction (AP-PCR), Amplified Fragment LengthPolymorphisms (AFLPs), and Single Nucleotide Polymorphisms (SNPs).

B. Plants Derived from a Plant of the Invention by Genetic Engineering

Many useful traits that can be introduced by backcrossing, as well asdirectly into a plant, are those, which may be introduced by genetictransformation techniques. Genetic transformation may therefore be usedto insert a selected transgene into a Brassica plant of the invention ormay, alternatively, be used for the preparation of transgenes, which canbe introduced by backcrossing. Methods for the transformation of plants,including Brassica, are well known to those of skill in the art.

Vectors used for the transformation of plant cells are not limited solong as the vector can express an inserted DNA in the cells. Forexample, vectors comprising promoters for constitutive gene expressionin Brassica cells (e.g., cauliflower mosaic virus 35S promoter) andpromoters inducible by exogenous stimuli can be used. Examples ofsuitable vectors include pBI binary vector. The “Brassica cell” intowhich the vector is to be introduced includes various forms of Brassicacells, such as cultured cell suspensions, protoplasts, leaf sections,and callus.

A vector can be introduced into Brassica cells by known methods, such asthe polyethylene glycol method, polycation method, electroporation,Agrobacterium-mediated transfer, particle bombardment and direct DNAuptake by protoplasts.

To effect transformation by electroporation, one may employ eitherfriable tissues, such as a suspension culture of cells or embryogeniccallus or alternatively one may transform immature embryos or otherorganized tissue directly. In this technique, one would partiallydegrade the cell walls of the chosen cells by exposing them topectin-degrading enzymes (pectolyases) or mechanically wound tissues ina controlled manner.

One efficient method for delivering transforming DNA segments to plantcells is microprojectile bombardment. In this method, particles arecoated with nucleic acids and delivered into cells by a propellingforce. Exemplary particles include those comprised of tungsten,platinum, and preferably, gold. For the bombardment, cells in suspensionare concentrated on filters or solid culture medium. Alternatively,immature embryos or other target cells may be arranged on solid culturemedium. The cells to be bombarded can be positioned at an appropriatedistance below the macroprojectile stopping plate. Microprojectilebombardment techniques are widely applicable, and may be used totransform virtually any plant species.

Agrobacterium-mediated transfer is another widely applicable system forintroducing gene loci into plant cells. An advantage of the technique isthat DNA can be introduced into whole plant tissues, thereby bypassingthe need for regeneration of an intact plant from a protoplast. ModernAgrobacterium transformation vectors are capable of replication in E.coli as well as Agrobacterium (and other Rhizobia), allowing forconvenient manipulations. Moreover, recent technological advances invectors for Agrobacterium-mediated gene transfer have improved thearrangement of genes and restriction sites in the vectors to facilitatethe construction of vectors capable of expressing various polypeptidecoding genes. The vectors described have convenient multi-linker regionsflanked by a promoter and a polyadenylation site for direct expressionof inserted polypeptide coding genes. Additionally, Agrobacteriumcontaining both armed and disarmed Ti genes can be used fortransformation.

In those plant strains where Agrobacterium-mediated transformation isefficient, it is the method of choice because of the facile and definednature of the gene locus transfer. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art (U.S. Pat. No. 5,563,055). For example, U.S. Pat. No.5,349,124 describes a method of transforming plant cells usingAgrobacterium-mediated transformation. By inserting a chimeric genehaving a DNA coding sequence encoding for the full-length B.t. toxinprotein that expresses a protein toxic toward Lepidopteran larvae, thismethodology resulted in plants having resistance to such insects.

A number of promoters have utility for plant gene expression for anygene of interest including but not limited to selectable markers,scorable markers, genes for pest tolerance, disease resistance,nutritional enhancements and any other gene of agronomic interest.Examples of constitutive promoters useful for Brassica plant geneexpression include, but are not limited to, the cauliflower mosaic virus(CaMV) P-35S promoter, which confers constitutive, high-level expressionin most plant tissues, including monocots; a tandemly duplicated versionof the CaMV 35S promoter, the enhanced 35S promoter (P-e35S) thenopaline synthase promoter, the octopine synthase promoter; and thefigwort mosaic virus (P-FMV) promoter as described in U.S. Pat. No.5,378,619 and an enhanced version of the FMV promoter (P-eFMV) where thepromoter sequence of P-FMV is duplicated in tandem, the cauliflowermosaic virus 19S promoter, a sugarcane bacilliform virus promoter, acommelina yellow mottle virus promoter, and other plant DNA viruspromoters known to express in plant cells.

Exemplary nucleic acids which may be introduced to the plants of thisinvention include, for example, DNA sequences or genes from anotherspecies, or even genes or sequences which originate with or are presentin the same species, but are incorporated into recipient cells bygenetic engineering methods rather than classical reproduction orbreeding techniques. However, the term “exogenous” is also intended torefer to genes that are not normally present in the cell beingtransformed, or perhaps simply not present in the form, structure, etc.,as found in the transforming DNA segment or gene, or genes which arenormally present and that one desires to express in a manner thatdiffers from the natural expression pattern, e.g., to over-express.Thus, the term “exogenous” gene or DNA is intended to refer to any geneor DNA segment that is introduced into a recipient cell, regardless ofwhether a similar gene may already be present in such a cell. The typeof DNA included in the exogenous DNA can include DNA which is alreadypresent in the plant cell, DNA from another plant, DNA from a differentorganism, or a DNA generated externally, such as a DNA sequencecontaining an antisense message of a gene, or a DNA sequence encoding asynthetic or modified version of a gene.

Many hundreds if not thousands of different genes are known and couldpotentially be introduced into a Brassica plant according to theinvention. Non-limiting examples of particular genes and correspondingphenotypes one may choose to introduce into a Brassica plant include oneor more genes for insect tolerance, such as a Bacillus thuringiensis(B.t.) gene, pest tolerance such as genes for fungal disease control,herbicide tolerance such as genes conferring glypho sate tolerance, andgenes for quality improvements such as yield, nutritional enhancements,environmental or stress tolerances, or any desirable changes in plantphysiology, growth, development, morphology or plant product(s). Forexample, structural genes would include any gene that confers insecttolerance including but not limited to a Bacillus insect control proteingene as described in WO 99/31248, herein incorporated by reference inits entirety, U.S. Pat. No. 5,689,052, herein incorporated by referencein its entirety, U.S. Pat. Nos. 5,500,365 and 5,880,275, hereinincorporated by reference it their entirety. In another embodiment, thestructural gene can confer tolerance to the herbicide glyphosate asconferred by genes including, but not limited to Agrobacterium strainCP4 glyphosate resistant EPSPS gene (aroA:CP4) as described in U.S. Pat.No. 5,633,435, herein incorporated by reference in its entirety, orglyphosate oxidoreductase gene (GOX) as described in U.S. Pat. No.5,463,175, herein incorporated by reference in its entirety.

Alternatively, the DNA coding sequences can affect these phenotypes byencoding a non-translatable RNA molecule that causes the targetedinhibition of expression of an endogenous gene, for example viaantisense- or cosuppression-mediated mechanisms. The RNA could also be acatalytic RNA molecule (i.e., a ribozyme) engineered to cleave a desiredendogenous mRNA product. Thus, any gene which produces a protein or mRNAwhich expresses a phenotype or morphology change of interest is usefulfor the practice of the present invention.

D. Definitions

In the description and tables herein, a number of terms are used. Inorder to provide a clear and consistent understanding of thespecification and claims, the following definitions are provided:

Allele: Any of one or more alternative forms of a gene locus, all ofwhich alleles relate to one trait or characteristic. In a diploid cellor organism, the two alleles of a given gene occupy corresponding locion a pair of homologous chromosomes.

Backcrossing: A process in which a breeder repeatedly crosses hybridprogeny, for example a first generation hybrid (F₁), back to one of theparents of the hybrid progeny. Backcrossing can be used to introduce oneor more single locus conversions from one genetic background intoanother.

Cultivated Variety: A Brassica oleracea variety which is suitable forconsumption and meets the requirements for commercial cultivation. Anexample is a broccoli variety. In addition to the plants themselves, andthe parts thereof suitable for consumption, such as the heads or leaves,the invention comprises parts or derivatives of the plant suitable forpropagation. Examples of parts suitable for propagation are organtissues, such as leaves, stems, roots, shoots and the like, protoplasts,somatic embryos, anthers, petioles, cells in culture and the like.Derivatives suitable for propagation are for instance seeds. The plantsaccording to the invention can be cultivated or propagated in theconventional manner but also by means of tissue culture techniques fromplant parts.

Crossing: The mating of two parent plants.

Cross-pollination: Fertilization by the union of two gametes fromdifferent plants.

Diploid: A cell or organism having two sets of chromosomes.

Enzymes: Molecules which can act as catalysts in biological reactions.

F₁ Hybrid: The first generation progeny of the cross of two nonisogenicplants.

Genotype: The genetic constitution of a cell or organism.

Haploid: A cell or organism having one set of the two sets ofchromosomes in a diploid.

Linkage: A phenomenon wherein alleles on the same chromosome tend tosegregate together more often than expected by chance if theirtransmission was independent.

Marker: A readily detectable phenotype, preferably inherited inco-dominant fashion (both alleles at a locus in a diploid heterozygoteare readily detectable), with no environmental variance component, i.e.,heritability of 1.

Phenotype: The detectable characteristics of a cell or organism, whichcharacteristics are the manifestation of gene expression.

Quantitative Trait Loci (QTL): Quantitative trait loci (QTL) refer togenetic loci that control to some degree numerically representabletraits that are usually continuously distributed.

Recombination event is understood to mean a meiotic crossing-over.

Regeneration: The development of a plant from tissue culture.

Self-pollination: The transfer of pollen from the anther to the stigmaof the same plant.

Single Locus Converted (Conversion) Plant: Plants which are developed bya plant breeding technique called backcrossing, wherein essentially allof the desired morphological and physiological characteristics of abroccoli variety are recovered in addition to the characteristics of thesingle locus transferred into the variety via the backcrossing techniqueand/or by genetic transformation.

Substantially Equivalent: A characteristic that, when compared, does notshow a statistically significant difference (e.g., p=0.05) from themean.

Tissue Culture: A composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant.

Transgene: A genetic locus comprising a sequence which has beenintroduced into the genome of a broccoli plant by transformation.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the invention, as limited only bythe scope of the appended claims.

All references cited herein are hereby expressly incorporated herein byreference.

E. Deposit Information

A deposit was made of at least 2500 seeds of broccoli hybrid RX05991199, which comprises a reduced introgression comprising a Myb28allele from Brassica villosa and an ELONG allele from Brassica oleracea,as described herein. The deposit was made with the American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209USA. The deposit is assigned ATCC Accession No. PTA-13165. The date ofdeposit was Aug. 24, 2012. Access to the deposits will be availableduring the pendency of the application to persons entitled thereto uponrequest. The deposits will be maintained in the ATCC Depository, whichis a public depository, for a period of 30 years, or 5 years after themost recent request, or for the enforceable life of the patent,whichever is longer, and will be replaced if nonviable during thatperiod. Applicant does not waive any infringement of their rightsgranted under this patent or any other form of variety protection,including the Plant Variety Protection Act (7 U.S.C. 2321 et seq.).

EXAMPLES Example 1 Development of Parent Lines with Improved MSBProfiles

One benefit of the current invention is that parent lines may be createdcomprising a reduced introgression of the invention, wherein the linesand hybrids derived therefrom exhibit an increased proportion of MSB/MSPand/or a greater stability of expression of MSB. The development of sucha broccoli line comprising a Myb28/ELONG reduced introgression can besummarized as follows:

The line FT69, which has elevated levels of the phytochemical MSP(glucoiberin) as a result of the presence of Myb28 and ELONG loci fromB. villosa (Mithen et al., 2003; Theor. Appl. Genet., 106:727-734), wascrossed with a breeding line designated BR9. The resulting progeny wascrossed with the male parent from Ironman (BRM 56-3905 SI). F1 progenywere grown in replications in a Wageningen selection trial. From plotsdesignated 408, 409, 410, 411 and 412, 89 plants were selected that wereanalyzed for glucoiberin (MSP) and glucoraphanin (MSB). Six plants withthe highest MSB were selected.

All 6 plants were selfed and backcrossed with the recurrent parent BRM56-3905 SI. The 6 selfings and 6 BC1 were planted in replicatedselection trials in Wageningen. Each BC1 (where BC=backcross) had 2plots with 24 plants/plot. From these 12 plots, a total of 84 plantswere selected that most resembled the recurrent parent. Heads from theseselections were sent for MSP and MSB analyses. Seven plants with highestMSB were kept for further selfing and BC. All 7 BC1 plants were selfedand backcrossed with the recurrent parent (BRM 56-3905 SI). Selfings and5 BC2 populations were sown and selected in replicated Wageningen Autumnselection trials. A total of 73 plants that resembled the recurrentparent most were selected and sent for MSP/MSB analyses. Eight BC2plants with highest MSB levels were kept for further selfing and BC.

All 8 BC2 plants were selfed and backcrossed with the recurrent parent(BRM 56-3905 SI). Four BC3 were obtained and planted in 1-4 replicationsin Wageningen. 47 plants closest to the recurrent parent were sent forMSP/MSB analyses. 7 BC3 plants with highest MSB levels were kept forfurther selfing and BC. All 7 BC3 plants were selfed and backcrossedwith the recurrent parent (BRM 56-3905 SI). 6 BC4 were obtained andthese were planted in selection trials. 54 plants closest to therecurrent parent were sent for MSP/MSB analyses. 8 BC4 plants with thehighest MSB levels were kept for further selfing and BC.

All 8 BC4 plants were selfed. 7 BC4 plants produced selfed seed and theBC4F2 were planted in unreplicated Wageningen selection trials. 37 BC4F2plants closest to the recurrent parent were selected and these were sentfor MSP/MSB analyses. 4 BC4F2 plants with highest MSB levels were keptfor further selfing and BC.

All 4 BC4F2 plants were selfed. All 4 produced selfed seed and the 4BC4F3 were planted in unreplicated selection trials. 12 BC4F3 plantsclosest to the recurrent parent were selected and sent for MSP/MSBanalyses. 4 BC4F2 plants with the highest MSB levels were kept forfurther selfing and BC. All selected plants from plot 1169 (570114) hadequally high MSB level of average 2.3 mmol/kg, or about 5× the level ofthe standard reference variety (General). This indicated that the sourcehas alleles fixed for high MSB.

All 3 selections from 1169 (BC4F3) were selfed. All 3 produced seed andthese were planted in selection trials. SNP13-580333 (1169-1, A75-1) wasthe most uniform on type and 5 plants were selected for MSB check. All 5had equally high levels of MSB of about 3.2 mmol/kg. 1169-1, alsodesignated BRM 53-3934 SI, was put on tissue culture for increase anduse as a male parent to RX-1199 (RX 05991199). An analysis of BRM53-3934 SI revealed that it contained the Myb28 reduced introgression,which results in a parent line with the ability to consistently produceprogeny with glucosinolates comprising an increased proportion ofMSB/MSP relative to plants having the B. villosa ELONG locus, as shownbelow.

Example 2 Analysis of Hybrid Broccoli Lines Having ElevatedGlucoraphanin

Self and outcrosses were made using different standard broccoli lines asfemale parents as shown in Table 1 in crosses with the following: (1)FT69 (high glucoiberin line with ELONG and Myb28 from B. villosa), (2)SNP13-580333 (high glucoraphanin line with ELONG from B. oleracea andMyb28 from B. villosa, as described above), and (3) SNP88-BRM51-1210(high glucoiberin line ELONG and Myb28 from B. villosa). As can be seen,regular broccoli lines have a relatively low amount of totalglucosinolates. The ratio between MSB and MSP is particularly shown tobe depending on the regular broccoli female line in the case of FT69(428-11-69), which comprises ELONG and Myb28 alleles from B. villosa. Incontrast, in all the crosses with SNP13-580333 most of theglucosinolates are glucoraphanin. This indicates the benefit of thereduced introgression in consistently producing hybrid varietiescomprising elevated glucoraphanin in crosses with multiple differentsecond parents.

TABLE 1 Levels of Glucoraphanin Resulting From Different Crosses Femaleline (regular Male self (self or crosses with different GlucoiberinGlucoraphanin broccoli line) high glucosinolates sources)micromoles/gramDWt micromoles/gramDWt Ratio MSB/MSP BR384-014 self 3.3118.74 5.66 BR384-014 FT69 (428-11-69) (contains Myb28 and 57.47 15.120.26 ELONG from B. villosa) BR384-014 SNP13-580333 (BRM 53-3934 SI) 3.7740.14 10.65 (contains reduced introgression) BR384-014 SNP88-BRM51-1210(contains Myb28 28.71 5.85 0.20 and ELONG from B. villosa) BR384-020Self 1.83 18.24 9.97 BR384-020 FT69 (428-11-69) 36.72 10.00 0.27BR384-020 SNP13-580333 2.34 26.29 11.23 BR384-020 SNP88-BRM51-1210 13.5333.96 2.51 BR384-024 self 1.59 3.64 2.29 BR384-024 FT69 (428-11-69)56.61 7.75 0.14 BR384-024 SNP13-580333 2.64 26.48 10.03 BR384-024SNP88-BRM51-1210 22.11 3.24 0.15 BR384-025 self 1.43 2.67 1.87 BR384-025FT69 (428-11-69) 39.66 6.39 0.16 BR384-025 SNP13-580333 3.52 27.55 7.83BR384-025 SNP88-BRM51-1210 14.25 9.59 0.67 BR384-026 self 1.74 5.08 2.92BR384-026 FT69 (428-11-69) 41.11 8.26 0.20 BR384-026 SNP13-580333 1.8030.53 16.93 BR384-026 SNP88-BRM51-1210 19.52 2.89 0.15 BR384-027 self2.06 13.52 6.55 BR384-027 FT69 (428-11-69) 9.32 47.34 5.08 BR384-027SNP13-580333 2.60 27.47 10.57 BR384-027 SNP88-BRM51-1210 6.61 28.12 4.26BR384-033 self 0.57 16.88 29.42 BR384-033 FT69 (428-11-69) 7.58 46.896.18 BR384-033 SNP13-580333 2.48 30.81 12.41 BR384-033 SNP88-BRM51-12105.23 28.75 5.49 BR384-040 self 0.91 14.03 15.46 BR384-040 FT69(428-11-69) 4.77 45.61 9.56 BR384-040 SNP13-580333 1.56 16.02 10.27BR384-040 SNP88-BRM51-1210 3.30 12.73 3.86 BR384-044 self 0.81 11.9414.74 BR384-044 FT69 (428-11-69) 9.48 50.08 5.28 BR384-044 SNP13-5803331.99 23.47 11.81 BR384-044 SNP88-BRM51-1210 5.19 27.95 5.38 BR384-048FT69 (428-11-69) 62.53 5.85 0.09 BR384-048 SNP13-580333 3.02 27.95 9.25BR384-048 SNP88-BRM51-1210 18.99 1.24 0.07 BR384-052 self 1.51 7.38 4.88BR384-052 FT69 (428-11-69) 8.27 38.54 4.66 BR384-052 SNP13-580333 2.4324.09 9.92 BR384-053 self 0.28 2.06 7.49 BR384-053 FT69 (428-11-69) 7.3053.95 7.39 BR384-053 SNP13-580333 0.64 21.64 33.74 BR384-053SNP88-BRM51-1210 3.39 14.50 4.27 BR384-057 self 1.43 3.79 2.64 BR384-057FT69 (428-11-69) 38.32 8.11 0.21 BR384-057 SNP13-580333 4.13 32.20 7.80BR384-057 SNP88-BRM51-1210 20.38 3.47 0.17 BR384-060 self 1.76 7.90 4.49BR384-060 FT69 (428-11-69) 10.62 42.68 4.02 BR384-060 SNP13-580333 2.7626.06 9.46 BR384-060 SNP88-BRM51-1210 5.46 23.19 4.25 BR384-061 self0.99 15.47 15.65 BR384-061 FT69 (428-11-69) 8.03 49.14 6.12 BR384-061SNP13-580333 2.58 35.61 13.82 BR384-061 SNP88-BRM51-1210 5.92 36.02 6.09BR384-069 self 1.63 7.02 4.32 BR384-069 FT69 (428-11-69) 9.33 56.09 6.01BR384-069 SNP13-580333 1.44 26.36 18.32 BR384-069 SNP88-BRM51-1210 4.6029.96 6.51 BR384-078 self 1.23 7.59 6.15 BR384-078 FT69 (428-11-69)10.54 36.99 3.51 BR384-078 SNP13-580333 2.75 24.94 9.06 BR384-078SNP88-BRM51-1210 5.74 23.42 4.08 BR384-082 self 1.57 10.41 6.64BR384-082 FT69 (428-11-69) 10.67 38.80 3.64 BR384-082 SNP13-580333 2.5329.25 11.55 BR384-082 SNP88-BRM51-1210 6.38 31.80 4.98 BR384-083 self1.31 8.44 6.45 BR384-083 FT69 (428-11-69) 9.33 39.32 4.21 BR384-083SNP13-580333 2.23 22.39 10.04 BR384-083 SNP88-BRM51-1210 4.85 21.90 4.52BR384-089 self 3.40 0.35 0.10 BR384-089 FT69 (428-11-69) 48.47 7.21 0.15BR384-089 SNP13-580333 4.67 24.59 5.27 BR384-089 SNP88-BRM51-1210 19.121.98 0.10

Example 3 Development of Broccoli Hybrids

As explained above, one embodiment of the invention comprises producingbroccoli hybrids wherein one or both parents of the hybrid comprise aMyb28 reduced introgression and, as a result, exhibit an elevated MSBcontent and/or more stable MSB content relative to hybrids lacking aparent comprising the introgression. One example of such a hybrid thatwas produced is the hybrid RX 05991199. This hybrid was created bycrossing parents BRM 53-3934 SI and BRM 56-3907 CMS, typically with BRM53-3934 SI as a male parent. The production of BRM 53-3934 SI isdescribed in Example 1 above. As explained, this parent contains theMyb28 reduced introgression. The female parent BRM 56-3907 CMS is aknown inbred that served as the parent of the commercial hybrid“Ironman.” BRM 56-3907 CMS is the subject of, and is described in, EUPlant Variety Rights Certificate #20341, granted Jun. 18, 2007.

A description of the physiological and morphological characteristics ofbroccoli hybrid RX 05991199 and the parent lines thereof is presentedbelow.

TABLE 2 Physiological and Morphological Characteristics of Hybrid RX05991199 Characteristic RX05991199CMS Ironman A Region of Adaptation NWEurope NW Europe B Maturity, Spring Planted days from direct seeding tono direct seeding no direct seeding 50% harvest days from transplantingto 50% 64 63 harvest transplant date 10-May-10 10-May-10 length ofharvest period in days  8  6 first harvest date 12-Jul-10 12-Jul-10 lastharvest date 19-Jul-10 17-Jul-10 harvest season (main crop at summersummer 50% harvest) time of harvest maturity (50% medium (Sumosun)medium (Sumosun) of plants) time of beginning of flowering medium*8(Coaster, medium*8 (Coaster, (50% of plants with at least Cruiser)Cruiser) 10% flowers) **choice for UPOV TG only C Seedling cotyledoncolor medium green medium green RHS color chart value for 147B 147Bseedling cotyledon color cotyledon anthocyanin intermediate intermediatehypocotyl anthocyanin strong strong D Plant plant height in centimeters 56.3 cm  61.0 cm from soil line to top of leaves head height incentimeters from  33.7 cm  34.7 cm soil line to top of leaves height atharvest maturity medium (Coaster) medium (Coaster) number of stems one(Ramoso Calabrese, one (Ramoso Shogun) Calabrese, Shogun) branchesmedium few habit intermediate intermediate market class fresh marketfresh market life cycle annual annual type of variety first generationhybrid first generation hybrid E Leaves outer leaves: number of leaves14 17 per plant (at harvest) outer leaves: width (at  22.6 cm  24.2 cmmidpoint of plant including petiole) leaf: width medium (Buccaneer,medium (Buccaneer, Green Belt) Green Belt) outer leaves: length (at 48.7 cm  53.2 cm midpoint of plant including petiole) leaf: length(including petiole) medium (Brigadeer, long (Green Duke, Sumosun) Laser)outer leaves: petiole length  22.1 cm  24.9 cm petiole: length medium(Emperor, long (Groene Ramoso Calabrese) Calabrese, Premium Crop) outerleaves: leaf ratio— 2:1 2:1 length/width outer leaves: leaf attachmentpetiolate petiolate outer leaves: wax presence weak intermediate leaf:number of lobes medium (Coaster, medium (Coaster, Topper) Topper) outerleaves: foliage color grey green grey green (with wax, if present) outerleaves: foliage color 189A 189A (with wax, if present; RHS color chartvalue) leaf blade: color grey green (Bishop) grey green (Bishop) leafblade: intensity of color dark dark leaf blade: anthocyanin absent(Claudia, absent (Claudia, coloration Embassy) Embassy) leaf blade:undulation of weak (Beaufort, Early weak (Beaufort, margin Pack, Laser,Paladin) Early Pack, Laser, Paladin) leaf blade: dentation of marginweak (Galaxy) weak (Galaxy) outer leaves: leaf shape elliptic ellipticouter leaves: leaf base blunt blunt outer leaves: leaf apex blunt bluntouter leaves: leaf margins slightly wavy slightly wavy outer leaves:leaf veins intermediate intermediate outer leaves: midrib slightlyraised not raised leaf blade: blistering medium (Medium Late medium(Medium 145, Skiff) Late 145, Skiff) outer leaves: attitude (leafsemi-erect (35-55 semi-erect (35-55 angle from ground) degrees) degrees)leaf: attitude (at beginning of semi-erect (Arcadia, semi-erect headformation) Asti, Civet, Claudia) outer leaves: torsion of leaf tip nonenone outer leaves: profile of upper planar planar side of leaf F Headlength of branching at base very short (Viola) very short (Viola)(excluding stem) diameter at widest point (at  15.2 cm  14.7 cm marketmaturity) depth (at market maturity)  9.7 cm  9.4 cm weight, markettrimmed (at 439.9 gm 400.3 gm market maturity) color grey green(Brigadeer, grey green Galaxy) (Brigadeer, Galaxy) intensity of colormedium medium RHS color chart value for head 189B 189B color anthocyanincoloration absent (Early White present (Brigadeer, Sprouting) Shogun,Viola) shape (at market maturity) transverse elliptic transverseelliptic (Buccaneer, Futura) (Buccaneer, Futura) dome shape (at marketdomed domed maturity) size (at market maturity) for medium** (Dundee,medium** (Dundee, US Exhibit C only **choice Early Man) Early Man)compactness/firmness (at medium (Late Corona) short pedicels/tight/market maturity) firm (Captain) surface knobbling (at market fine(Apollo, Brigadeer) medium (Southern maturity) Comet) texture medium(Clipper, fine (Auriga, Bishop, Coaster) Green Top) bead size (at marketmaturity) medium small flower buds (at market even in size even in sizematurity) anthocyanin coloration of leaf absent absent axils (at marketmaturity) anthocyanin coloration of leaf absent absent veins (at marketmaturity) anthocyanin coloration of leaf absent absent blade (at marketmaturity) anthocyanin coloration of absent absent entire plant (atmarket maturity) anthocyanin coloration of leaf absent (Claudia, absent(Claudia, petiole (at market maturity) Embassy) Embassy) color of headleaves (at market green green maturity) RHS color chart value for theN189B N189B color of head leaves bracts absent (Gem, Orion) absent (Gem,Orion) secondary heads (at market combination, present** present,combination maturity) (Marathon, Tribute, Late Purple Sprouting)prominence of secondary heads intermediate (Citation) weak (at marketmaturity) number of secondary heads (at  4  2 market maturity) G FlowerColor **choice for UPOV TG yellow** (Brigadeer, yellow** (Brigadeer,only Orion) Orion) intensity of yellow color medium (Capitol, medium(Capitol, Corvet) Corvet) color 4B 4B stalk color green green RHS colorchart value for 138B 138B flower stalk color male sterility present(Chevalier, present (Chevalier, Montop) Montop) *These are typicalvalues. Values may vary due to environment. Other values that aresubstantially equivalent are also within the scope of the invention.

TABLE 3 Physiological and Morphological Characteristics of Line BRM53-3934 SI Characteristic BMR53-3934SI Sibsey A Region of Adaptation NWEurope NW Europe B Maturity, Spring Planted days from direct seeding tono direct seeding no direct seeding 50% harvest days from transplantingto 50% 76 54 harvest transplant date 10-May-10 10-May-10 length ofharvest period in days 16  5 first harvest date 17-Jul-10 1-Jul-10 lastharvest date 1-Aug-10 5-Jul-10 harvest season (main crop at summersummer 50% harvest) time of harvest maturity (50% very late (Late Purplevery early of plants) Sprouting) (Earlyman, Primor) time of beginning offlowering late** (Shogun, Viola) early** (Clipper, (50% of plants withat least Southern Comet) 10% flowers) **choice for UPOV TG only CSeedling cotyledon color medium green medium green RHS color chart valuefor 147B 147B seedling cotyledon color cotyledon anthocyanin weakintermediate hypocotyl anthocyanin weak intermediate D Plant plantheight in centimeters  51.7 cm  55.0 cm from soil line to top of leaveshead height in centimeters from  30.3 cm  50.0 cm soil line to top ofleaves height at harvest maturity medium (Coaster) medium (Coaster)number of stems one (Ramoso Calabrese, one (Ramoso Shogun) Calabrese,Shogun) branches medium many habit compact compact market class freshmarket fresh market life cycle annual annual type of variety inbredfirst generation hybrid E Leaves outer leaves: number of leaves 20 12per plant (at harvest) outer leaves: width (at  21.8 cm  18.9 cmmidpoint of plant including petiole) leaf: width medium (Buccaneer,narrow (Arcadia, Green Belt) Brigadeer) outer leaves: length (at  45.3cm  39.1 cm midpoint of plant including petiole) leaf: length (includingpetiole) medium (Brigadeer, short (Dandy Early, Sumosun) Emperor) outerleaves: petiole length  20.2 cm  16.0 cm petiole: length medium(Emperor, short (High Sierra, Ramoso Calabrese) Padovano) outer leaves:leaf ratio— 2:1 2:1 length/width outer leaves: leaf attachment petiolatepetiolate outer leaves: wax presence intermediate intermediate leaf:number of lobes medium (Coaster, medium (Coaster, Topper) Topper) outerleaves: foliage color blue green grey green (with wax, if present) outerleaves: foliage color N198A 189A (with wax, if present; RHS color chartvalue) leaf blade: color blue green (Citation, grey green (Bishop)Esquire, Symphony) leaf blade: intensity of color medium medium leafblade: anthocyanin absent (Claudia, absent (Claudia, coloration Embassy)Embassy) leaf blade: undulation of weak (Beaufort, Early strong (Aikido,margin Pack, Laser, Paladin) Marathon, Samurai) leaf blade: dentation ofmargin weak (Galaxy) weak (Galaxy) outer leaves: leaf shape ellipticelliptic outer leaves: leaf base blunt blunt outer leaves: leaf apexblunt blunt outer leaves: leaf margins slightly wavy very wavy outerleaves: leaf veins intermediate thin outer leaves: midrib slightlyraised not raised leaf blade: blistering absent or very weak absent orvery weak (Buccaneer, Colibri) (Buccaneer, Colibri) outer leaves:attitude (leaf semi-erect (35-55 semi-erect (35-55 angle from ground)degrees) degrees) leaf: attitude (at beginning of semi-erect (Arcadia,semi-erect head formation) Asti, Civet, Claudia) outer leaves: torsionof leaf tip none weak outer leaves: profile of upper concave convex sideof leaf F Head length of branching at base short (Brigadeer, medium(Capitol, (excluding stem) Buccaneer Emperor) Green Duke, Perseus)diameter at widest point (at  11.4 cm  13.7 cm market maturity) depth(at market maturity)  10.4 cm  10.9 cm weight, market trimmed (at 164.3gm 182.7 gm market maturity) color light green; grey-green grey-green(Brigadeer, Galaxy) (Brigadeer, Galaxy) intensity of color medium mediumRHS color chart value for head 189A/144A N189B color anthocyanincoloration absent (Early White absent (Early White Sprouting) Sprouting)shape (at market maturity) circular (Esquire) transverse elliptic(Buccaneer, Futura) dome shape (at market domed domed maturity) size (atmarket maturity) for US very small (Early Purple small** (Orbit, ExhibitC only **choice Sprouting) Scorpio) compactness/firmness (at longpedicels/loose medium (Late market maturity) (Caravel) Corona) surfaceknobbling (at market medium (Southern fine (Apollo, maturity) Comet)Brigadeer) texture fine (Auriga, Bishop, fine (Auriga, Bishop, GreenTop); coarse Green Top) (Citation) bead size (at market maturity)small/medium/large small flower buds (at market uneven in size (cateye)even in size maturity) anthocyanin coloration of leaf absent absentaxils (at market maturity) anthocyanin coloration of leaf absent absentveins (at market maturity) anthocyanin coloration of leaf absent absentblade (at market maturity) anthocyanin coloration of entire absentabsent plant (at market maturity) anthocyanin coloration of leaf absent(Claudia, absent (Claudia, petiole (at market maturity) Embassy)Embassy) color of head leaves (at market green green maturity) RHS colorchart value for the N189B 189A color of head leaves bracts present(Ramoso absent (Gem, Orion) Calabrese) secondary heads (at market basal,present** combination maturity) (Marathon, Tribute, Late PurpleSprouting) prominence of secondary heads intermediate (Citation)intermediate (at market maturity) (Citation) number of secondary heads(at  3  4 market maturity) G Flower Color **choice for UPOV TG yellow**(Brigadeer, yellow** (Brigadeer, only Orion) Orion) intensity of yellowcolor dark (Gem, Orion) dark (Gem, Orion) color 4A 4A stalk color greengreen RHS color chart value for 138B 138B flower stalk color malesterility absent (Marathon) present (Chevalier, Montop) *These aretypical values. Values may vary due to environment. Other values thatare substantially equivalent are also within the scope of the invention.

The MSB content of hybrid RX 05991199 relative to the hybrid “Heritage”was the subject of an objective analysis. The results of the analysisare presented below.

TABLE 4 Analysis of MSB Content of RX 05991199 Varieties Least-SquaresMeans for MSB (micromoles/gm/FW) RX 05991199 3.5833927 Heritage1.6311887

A head-to-head analysis was also made of glucoraphanin (MSB) content ofRX 05991199 relative to the hybrid Ironman in multiple locations andplantings. The results are presented below.

TABLE 5 Analysis of Glucoraphanin Content of RX 05991199 Relative toHybrid calculated in μmol per gDW Gluco- Gluco- Variety CountrySubRegion Planting iberin raphanin Ironman Italy South A 2.1 3.9 RX05991199 Italy South A 5.0 20.0 RX 05991199 Italy South A 1.5 6.7Ironman Spain South B 2.1 3.1 RX 05991199 Spain South B 3.5 13.4 IronmanItaly South C 3.2 9.2 Ironman Italy South C 3.6 9.1 Ironman Italy SouthC 3.4 10.3 Ironman Italy South C 3.3 10.8 RX 05991199 Italy South C 4.720.7 RX 05991199 Italy South C 5.4 22.3 RX 05991199 Italy South C 5.526.0 RX 05991199 Italy South C 4.7 22.9 Ironman Spain South D 1.9 5.5Ironman Spain South D 2.8 9.6 Ironman Spain South D 2.4 5.9 RX 05991199Spain South D 5.1 21.7 RX 05991199 Spain South D 5.5 24.8 RX 05991199Spain South D 5.1 25.7 Ironman UK North A 2.5 9.8 Ironman UK North A 3.112.8 RX 05991199 UK North A 3.8 21.3 RX 05991199 UK North A 3.4 24.4Ironman UK North B 0.9 7.1 Ironman UK North B 1.9 12.1 RX 05991199 UKNorth B 2.4 11.9 RX 05991199 UK North B 3.0 16.0 Ironman UK North C 1.85.8 Ironman UK North C 1.9 5.8 RX 05991199 UK North C 3.1 14.1 RX05991199 UK North C 3.3 15.1 Ironman UK North D 1.1 6.2 Ironman UK NorthD 0.9 6.2 RX 05991199 UK North D 3.2 17.2 RX 05991199 UK North D 3.116.6 ironman Italy South F 0.9 5.1 ironman Italy South F 1.0 5.3 RX05991199 Italy South F 3.8 24.5 RX 05991199 Italy South F 3.6 25.1ironman Italy South A 0.9 4.3 ironman Italy South A 1.0 5.1 RX 05991199Italy South A 4.6 23.5 RX 05991199 Italy South A 4.0 22.4 ironman SpainSouth B 1.5 6.0 ironman Spain South B 2.4 11.2 RX 05991199 Spain South B4.0 20.7 RX 05991199 Spain South B 4.4 22.1 ironman Spain South C 1.98.3 ironman Spain South C 1.7 8.8 RX 05991199 Spain South C 4.1 24.3 RX05991199 Spain South C 4.3 22.4 Ironman Spain South D 1.2 5.5 IronmanSpain South D 0.9 4.9 RX 05991199 Spain South D 3.5 19.0 RX 05991199Spain South D 2.8 17.5 Ironman Italy South E 0.9 7.6 Ironman Italy SouthE 0.8 6.7 RX 05991199 Italy South E 3.0 24.4 RX 05991199 Italy South E1.8 23.3 Ironman Spain South F 0.8 5.0 Ironman Spain South F 0.6 3.8 RX05991199 Spain South F 2.9 17.1 RX 05991199 Spain South F 3.0 19.3Ironman Spain South G 1.0 6.3 Ironman Spain South G 0.9 6.2 RX 05991199Spain South G 3.4 20.8 RX 05991199 Spain South G 3.0 19.9 Ironman SpainSouth H 2.5 14.7 Ironman Spain South H 2.3 14.1 RX 05991199 Spain SouthH 3.6 21.9 RX 05991199 Spain South H 4.0 23.0 Ironman UK North B 0.9 6.2Ironman UK North B 1.0 6.1 RX 05991199 UK North B 4.2 20.9 RX 05991199UK North B 3.6 18.6 Ironman UK North C 2.1 6.3 Ironman UK North C 1.96.4 RX 05991199 UK North C 4.7 15.1 RX 05991199 UK North C 4.4 15.5Ironman UK North D 1.7 11.3 Ironman UK North D 1.1 6.3 RX 05991199 UKNorth D 4.1 22.0 RX 05991199 UK North D 2.7 14.6 Ironman UK North E 2.111.9 RX 05991199 UK North E 5.3 24.2 Ironman UK North F 1.2 8.0 IronmanUK North F 1.5 8.7 RX 05991199 UK North F 4.2 19.9 RX 05991199 UK NorthF 4.5 21.9 Ironman UK North G 1.0 6.1 Ironman UK North G 1.0 6.7 RX05991199 UK North G 2.7 16.8 RX 05991199 UK North G 2.8 17.7 Ironman UKNorth H 1.0 6.7 Ironman UK North H 0.8 5.8 RX 05991199 UK North H 3.419.2 RX 05991199 UK North H 3.9 23.1 Ironman Spain South A 0.6 4.0Ironman Spain South A 0.9 4.2 Ironman Spain South A 0.8 3.7 RX 05991199Spain South A 2.6 13.3 RX 05991199 Spain South A 2.7 14.1 RX 05991199Spain South A 2.8 13.4 Ironman Italy South B 1.4 7.3 Ironman Italy SouthB 1.2 8.2 RX 05991199 Italy South B 4.9 26.6 RX 05991199 Italy South B3.5 18.7 Ironman Spain South C 1.1 5.9 Ironman Spain South C 1.0 4.8Ironman Spain South C 1.0 5.5 RX 05991199 Spain South C 3.3 16.0 RX05991199 Spain South C 2.7 13.0 RX 05991199 Spain South C 2.9 11.7Ironman Italy South D 1.1 5.9 Ironman Italy South D 1.1 5.3 RX 05991199Italy South D 2.9 16.0 RX 05991199 Italy South D 3.9 21.4 Ironman SpainSouth E 0.9 4.3 Ironman Spain South E 1.0 3.9 Ironman Spain South E 1.14.8 RX 05991199 Spain South E 3.6 21.2 RX 05991199 Spain South E 2.216.5 RX 05991199 Spain South E 2.5 15.5 Ironman Spain South F 1.1 4.6Ironman Spain South F 0.8 4.4 Ironman Spain South F 1.0 4.5 RX 05991199Spain South F 1.5 9.7 RX 05991199 Spain South F 2.3 15.4 RX 05991199Spain South F 2.4 15.3 Ironman Spain South G 1.2 4.4 Ironman Spain SouthG 1.4 5.6 Ironman Spain South G 1.0 3.4 RX 05991199 Spain South G 2.916.7 RX 05991199 Spain South G 2.8 18.7 RX 05991199 Spain South G 3.015.9 Ironman UK North L 0.7 8.2 Ironman UK North L 0.8 9.8 Ironman UKNorth L 0.8 10.3 RX 05991199 UK North L 1.6 15.0 RX 05991199 UK North L1.9 17.1 RX 05991199 UK North L 1.9 18.1 Ironman UK North M 0.2 4.9Ironman UK North M 0.3 4.5 Ironman UK North M 0.3 5.2 RX 05991199 UKNorth M 1.1 8.5 RX 05991199 UK North M 1.6 9.8 RX 05991199 UK North M1.3 10.0

Example 4 Use of Genetic Markers to Identify and Track ReducedIntrogressions

Genetic marker assays were developed to genotype for Myb28 and ELONGalleles. The assays thus permit identification of a reducedintrogression in accordance with the invention as well as markerassisted introduction of the reduced introgression into any othergenotype.

A. Markers for Detection of ELONG

A marker designated QTL1-BoGLS-ELONG was developed and permits detectionof the presence or absence of a B. villosa ELONG allele. This marker canbe detected using the primer pair AF399834F2: 5′-cggattttcaaattttctcg-3′(SEQ ID NO:1) and AF399834R2: 5′-atttcgcatgaccactaggc-3′ (SEQ ID NO:2).To detect the marker, plates were loaded with 20 ng DNA template(sample) in a 2 μL volume. 34, master mix (0.437 μL water, 2.5 μL Q PCR(ROX) mix, 0.063 uL assay mix) was added to each well for a final volumeof 5 μL. PCR conditions were as follows: 95° C. for 15 min then 40cycles of 95° C. for 15 sec, 60° C. for 1 min. FIG. 4 shows results ofan assay for different ELONG alleles using the marker. The V allele isindicative of the B. villosa ELONG allele, while A, B and C alleles areexamples of alleles found in broccoli without a B. villosa ELONG allele.

B. Marker for the Detection of Myb28

The identification of genetic polymorphisms in Myb28 alleles and theiruse as genetic markers is described in U.S. Provisional Appln. Ser. No.61/700,731, filed concurrently herewith, the disclosure of which isincorporated herein by reference in its entirety. Specifically, sequencealignments were described therein between B. oleracea alleles and thecorresponding Myb28 sequences to identify polymorphisms that can be usedfor marker based selection for a Myb28 allele of choice. The results areshown in FIG. 5, which is an alignment between a consensus sequence ofthe Myb28 locus from B. villosa contained in broccoli variety FT69, anda consensus sequence of the corresponding locus from broccoli withoutincreased level of glucosinolate, e.g. B. oleracea, (Oleracea). Shownare 26 polymorphisms (e.g. single feature polymorphisms (SFPs)—of whichthere are 16 SNPs and 10 indels) detected in a sequence with a totallength of 2202 bp. Any of these or other identified polymorphisms may beused as genetic markers for the presence or absence of a desired Myb28,including from B. oleracea or B. villosa.

A TaqMan assay (NBOLI009111370) was designed based on one of thesequence polymorphisms identified, as follows:

NBOLI009111370 sequence (SEQ ID NO: 3):GACCACCTAAAGACAAGAATAGTGAAAGAGATAAGATGGAAGACCAAAGTTAATCAAATTTATTTTGAAGCTTTT[C/T]TATGGAATAGAGACTAAAATGATGTGTGCTATTGCAATTTTTAGTCACATATTGCTAATCAAACACATATTTTGCATCAGAGAATTGTCAAATACATGAAAAAAATAAAGAATAATTTTTForward primer (SEQ ID NO: 4): GTGAAAGAGATAAGATGGAAGACCAAAGTReverse primer(SEQ ID NO: 5): GTGACTAAAAATTGCAATAGCACACATCAVic probe (SEQ ID NO: 6): CTATTCCATAGAAAAGC Fam probe (SEQ ID NO: 7):CTATTCCATAAAAAAGC

The assay was carried out using standard procedures as follows: Loadplates with 20 ng DNA template in 5 μL volume. Add 10 μL master mix (2parts each of 1×PCR mix, 0.437 μL water, 2.5 μL Q PCR (ROX) mix, 0.063μL assay mix, 2 μL primers at 5 ng/μL) to each well for a final volumeof 15 μL.

PCR conditions are as follows: 50° C. for 2 min followed by 95° C. for 2min then 40 cycles of 95° C. for 15 sec, 60° C. for 1 min.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

1-29. (canceled)
 30. A composition prepared from a Brassica oleraceaplant with elevated glucosinolate content, or a part thereof, whereinthe plant or part thereof comprises a chromosomal segment comprising aMyb28 allele from Brassica villosa and an ELONG allele from Brassicaoleracea, wherein a sample of seed comprising said Myb28 allele and saidELONG allele was deposited under ATCC Accession No. PTA-13165.
 31. Thecomposition of claim 30, wherein the part thereof is defined as afloret, a seed, a cell, a root, a stem, a shoot, a leaf, a head, aflower, an anther, a petiole, pollen, a microspore, a haploid embryo, asomatic embryo, or a callus.
 32. The composition of claim 30, whereinthe plant is a broccoli plant.
 33. The composition of claim 30, whereina sample of seed comprising said chromosomal segment was deposited underATCC Accession No. PTA-13165.
 34. The composition of claim 30, whereinthe plant is inbred.
 35. The composition of claim 30, wherein the plantis hybrid.
 36. The composition of claim 30, wherein the plant ishomozygous for said Myb28 allele from Brassica villosa.
 37. Thecomposition of claim 30, wherein the plant is heterozygous for saidMyb28 allele from Brassica villosa.